The innate immune system senses non-self nucleic acids via germ-line encoded pattern recognition receptors. RNA is sensed via Toll-like receptor (TLR)-3, -7 and -8 or by the cytoplasmic RNA helicases RIG-I and MDA-5. Little is known about sensors for cytoplasmic DNA, which when delivered to the cytoplasm trigger type I IFN gene transcription and caspase-1-mediated processing of IL-12. We have identified PISA (PYHIN protein stimulating ASC), one of four human PYHIN proteins as a receptor for cytosolic DNA. The HIN200 domain of PISA binds to DNA, while the PYD domain (but not that of PYHIN1-3) associates with the adapter molecule ASC to activate NF-:B and caspase-1. Knockdown of PISA downregulates caspase-1-mediated IL-12 responses, with concomitant increases in type I interferon production. Our hypothesis is that PISA serves to recognize microbial cytosolic DNA (such as bacterial or viral DNA) and that PISA induces pro-inflammatory responses towards cytoplasmic DNA via the assembly of an inflammasome that engages ASC and caspase-1. In this study, we propose to further characterize the role of PISA in the recognition of viral and bacterial cytosolic DNA. We will also generate a PISA knock-out mouse by gene-trap technology and/or targeted gene deletion and propose to analyze the in vivo relevance of PISA for microbial infections. Finally, we will assess the role of PISA for the modulation of interferon responses towards cytoplasmic DNA. Elucidation of the molecular mechanisms of cytosolic DNA recognition could lead to novel therapeutic targets for microbial infections and thereby enable novel strategies that could prevent inflammatory reactions to gene therapy approaches. Public Health Relevance: Pathogens are detected by receptors expressed in different areas of cells of the immune system. This grant is focused on understanding how a cytosolic protein, which we have identified called PISA (PYHIN protein stimulating ASC) senses pathogens by virtue of their DNA. Elucidation of molecular details of PISA activation could lead to novel strategies of pharmacological interference for diseases based on intracellular DNA recognition.